Folia Cryptog. Estonica, Fasc.

49: 0–0 (2012)

First records of two ascomycetes on Phleum pretense in Estonia

Triin Varvas & Bellis Kullman

Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences,
Kreutzwaldi 5, 51014 Tartu, Estonia.
E-mails: triin.varvas@emu.ee, bellis.kullman@emu.ee

Abstract: Within a study on endophytic fungi of Timothygrass (Phleum pratense), two ascomycetes were recorded for the
first time in Estonia. Based on morphological and molecular methods, the species were determined as Alternaria arbusti and
A. viburni (teleomorph Lewia viburni), new to P. pratense.

Kokkuvõte: Kahe uue põldtimuti kottseene esmaleiud Eestis

Uurides põldtimuti (Phleum pratense) endofüütseid seeni leiti Eestile kaks uut kottseene liiki. Kasutades puhaskultuuri ja
molekulaarseid tunnuseid määrati liigid Alternaria arbusti ja A. viburni (teleomorf Lewia viburni), mis on uued mikroseened
põldtimutile.

INTRODUCTION
The important and widespread fungal genus Al-
ternaria was originally described by Nees in 1816
(Simmons, 2002); it comprises ca 300 species of
fungi commonly isolated from plants, soil, food,
and air (Kirk et al., 2008). Prior to our study,
only 14 species of this genus had been recorded
in Estonia, according to eElurikkus database
(http://elurikkus.ut.ee/). The genus includes
many species that produce mycotoxins harmful
to their plant hosts as well as to humans and
animals (Simmons, 2007; Agrios, 2005; Chris-
tensen, 2005). The total losses caused by the
species of this genus are estimated to be among
the highest brought about by any plant pathogen
(Agrios, 2005). The species of this anamorphic
genus cause serious problems in agriculture
by reducing crop yield and causing spoilage in
storage (Andersen et al., 2001), but many spe-
cies also produce secondary metabolites that
are used as biocontrol agents (Yandco-Ables et
al., 2006; Mohan Babu et al., 2002).
The genus Alternaria comprises many species-
groups differing in several morphological char-
acters. A. arbusti and A.viburni belong to the A.
infectoria species-group, which is characterized
by the production of long secondary conidi-
ophores between conidia (Simmons, 2002). A.
arbusti is known as the causal agent of leaf le-
sions on Asian pear (Pyrus pyrifolia) (Simmons &
Roberts, 1993). The degree of the pathogenicity
of A. viburnum is unclear. The above species
have previously been identified only during
plant pathogen studies. We found A. arbusti
and A.viburni during a study on endophytic
fungi of Timothygrass. It has been reported that
microenvironmental conditions can change the
lifestyle of fungi from mutualistic to parasitic
(Kniskern & Rausher 2006, Johnson & Oelmül-
ler 2009). Considering the above circumstances,
the genus Alternaria deserves in-depth research
of both its ecology and toxicology. The purpose
of this paper was to give a brief overview of the
two fungal species, A. arbusti and A.viburni,
new to Estonia.
MATERIALS AND METHODS
Samples of Timothygrass (Phleum pratense L.)
were collected for endophyte studies in autumn
2008 from the FAHM (Free Air Humidity Ma-
nipulation) experimental plots, located in Rõka,
Tartu County, Estonia (58°14'44"N, 27°17'58"E).
Plants were processed for endophytes by
surface sterilization of the tissues and planting
the material on agar media. From plants, 5 × 5
mm fragments were cut and the surfaces were
disinfected by rinsing in 96% ethanol for 1 min,
followed by repeated rinsing in sterile water.
Fragments were cut into smaller sections and
placed in Petri dishes (9 cm diam.) on potato
dextrose agar (PDA, Merck KGaA Germany). The
subsequent pure fungal cultures were studied
2 Folia Cryptog. Estonica
macroscopically and microscopically using a
Zeiss Axioskop 40 FL microscope, AxioCam MRc
camera and the Axio Vison 1.6 program. Species
were determined using taxonomic monographs
and manuals by Domsch et al. (1980), Simmons
(2007) and Ellis & Ellis (1997).
For molecular identification, 7–10 days after
inoculation of subcultures, a small piece of my-
celium with agar was cut and preserved in so-
dium dodecyl sulphate (SDS). Genomic DNA was
extracted according to Gardes & Bruns (1993).
ITS regions of rDNA were amplified by PCR using
primers ITS1F (5´ to 3´: CTT GGT CAT TTA GAG
GAA GTA A) and ITS4 (5´ to 3´: TCC TCC GCT
TAT TGA TAT GC). PCR product was purified
with Exo-Sap (Fermentas, Lithuania) following
the manufacturer’s instructions. The samples
were sequenced in Macrogen (Korea) with prim-
ers ITS1 (5` to 3`: TCC GTA GGT GAA CCT GCG
G) and ITS5 (5` to 3`: GGA AGT AAA AGT CGT
AAC AAG G). The sequences were edited using
the program Sequence Scanner 1.0 (Applied
Biosystems, Foster City, California, USA) and
compared with the sequences in the GenBank
using the BLASTn algorithm. Sequences with
<97% ITS rDNA sequence similarity were con-
sidered homospecific. The voucher strain of A.
arbusti culture was deposited in the collection
of fungal living cultures [TFC 2010-011] at the
Estonian University of Life Sciences and the
sequences of both taxa were deposited in the
GenBank (accession no JN688919 A. arbusti,
JN688921 A. viburni).
TAXONOMIC DESCRIPTIONS
Alternaria arbusti E. G. Simmons, Mycotaxon
48: 103 (1993).
Teleomorph – Unknown.
Culture description – Colonies loosely woolly
with sparse greyish aerial hyphae. Colony colour
dark-grey to black or light-grey to dark brown
with a lighter border. Reverse side has either
concentric rings from light yellow to dark brown
or dark hypha radiating out from the centre (Fig.
1). Only juvenile club-shaped conidia observed,
forming chains or singly, divided by transverse
and longitudinal septations, 15–32 × 4–12 μm.
Conidia colour light to medium brown.
Substrata – P. pratense leaf (studied Estonian
material); originally isolated from leaves of Pyrus
pyrifolia (Simmons, 1993).
Holotype – BPI 802727.
Comments – The species was identified by mo-
lecular methods. It has earlier been recorded on
cherry fruit (Prunus sp.) as a nontoxigenic spe-
cies (Simmons, 1993). A study on the secondary
metabolites of the Alternaria species established
that A. arbusti does not produce a potential
phytotoxic compound present in some other
members of the A. infectoria species-group. At
the same time, it produces another biologically
active metabolite — infectopyrone (Christensen
et al., 2005).
Alternaria viburni E. G. Simmons, Mycotaxon
83: 132 (2002).
Teleomorph – Lewia viburni E.G. Simmons
& McKemy, in Simmons, Mycotaxon 83: 130
(2002) (see also a description of teleomorph
Simmons, 2007).
Culture description – Colony cottony with sparse
whitish aerial hypha. Colony colour dark-grey-
brownish to light-grey. Reverse side light grey
and smooth with some concentric rings. No co-
nidia observed. Culture was contaminated upon
re-culturing, pure culture or culture photo not
preserved. Substrata: P. pratense leaf (studied
Estonian material); originally isolated from
leaves of Viburnum sp. (Simmons, 2002).
Holotype – BPI 841915.
Comments – The species was identified by
molecular methods. This little known species
could have a great potential in the research
of secondary metabolites. A chemotaxonomic
marker study conducted on the A. infectoria
species-group by Christensen et al. (2005)
showed that A. viburni produced novae-zelandin
A and novae-zelandin B – secondary metabolites
with a chemical structure similar to that of some
phytotoxic compounds from other fungi.
A. arbusti and L. viburni are new endophytic
fungi on Phleum pratense. The species of the
genus Alternaria and even its strains can cause
diseases in different plants by producing distinct
host-specific toxins. In this case pathogenicity
only occurs in the presence of a specific toxin
(Markham & Hille, 2001). Considering this, the
toxin production of the strains isolated from
Phleum pratense should be determined for
pathogenicity.

Fig. 1. Two Alternaria arbusti isolates on PDA at 21 days.
ACKNOWLEDGEMENTS
We wish to express our thanks to K. Kasekamp
for performing molecular analyses and for coop-
eration. Financial support was received from the
Ministry of Education and Research of Estonia
(target financing SF0170057s09).
REFERENCES
Ables, C. Y., Rosskopf, E. N. & Charudattan, R. 2006.
Putting plant pathogens to work: progress and
possibilities in weed biocontrol part 2. Improving
weed control efficacy. APS Net Plant Pathology
Online. http://www.apsnet.org/online/feature/
weed2. Cited 07.05.2012
3
Agrios, G. N. 2005. Plant Pathology (5th edition).
Elsevier-Academic Press, San Diego, CA. 922 pp.
Andersen, B., Kroger, E. & Roberts, R. G. 2001. Chemi-
cal and morphological segregation of Alternaria
alternata, A. gaisen and A. longipes. Mycological
Research 105: 291–299.
Christensen, K. B., Van Klink, J. W., Weavers, R. T.,
Larsen, T. O., Andersen, B. & Phipps, R. K. 2005.
Novel chemotaxonomic markers of the Alternaria
infectoria species-group. Journal of Agricultural
and Food Chemistry 53(24): 9431–9435.
Domsch, K. H., Gams, W. & Anderson, T. H. 1980.
Compendium of soil fungi. IHW-Verlag, Eching,
Germany. 859 pp.
4 Folia Cryptog. Estonica
eElurikkus – http://elurikkus.ut.ee/ Cited 13.05.2012
Ellis, M. B. & Ellis, J. P. 1997. Microfungi on Land
Plants. An Identification Handbook. Richmond
Publishing, Slough. 868 pp.
Gardes, M. & Bruns, T. D. 1993. ITS primers with
enhanced specificity for basidiomycetes – applica-
tion to the identification of mycorrhizae and rusts.
Molecular Ecology 2: 113–118.
Johnson, J. M. & Oelmüller, R. 2009. Mutualism or
parasitism: life in an unstable continuum. What
can we learn from the mutualistic interaction
between Piriformospora indica and Arabidop-
sis thaliana? Endocytobiosis Cell Research 19:
81–111.
Kirk, P. M., Cannon, P. F., Minter, D. W. & Stalpers,
J. A. 2008. Ainsworth and Bisby’s Dictionary of
the Fungi. 10th ed, CABI Bioscience, UK. 784 pp.
Kniskern, J. M. & Rausher, M. D. 2006. Environ-
mental variation mediates the deleterious effects
of Coleosporium ipomoeae on Ipomoea purpurea.
Ecology 87: 675–685.
Markham, J. E. & Hille, J. 2001. Host-selective toxins
as agents of cell death in plant-fungus interac-
tions. Molecular Plant Pathology 2(4): 229–239.
Mohan Babu, R., Sajeena, A., Seetharaman, K., Vid-
hyasekaran, P., Rangasamy, P., Som Prakash, M.,
Senthil Raja, A. & Biji, K. R. 2002. Host range of
Alternaria alternate – a potential fungal biocontrol
agents for waterhyacinth in India. Crop Protection
21(10): 1083-1085.
Simmons, E. G. 1993. Alternaria themes and varia-
tions (63–72). Mycotaxon 48: 91–107.
Simmons, E. G. 2002. Alternaria themes and varia-
tions (305–309). Mycotaxon 83: 127–145.
Simmons, E. G. 2007. Alternaria: an identification
manual. APS Press. 775 pp.
Simmons, E. G. & Roberts, R. G. 1993. Alternaria
themes and variations (73). Mycotaxon 48:
109–140.